Methods of treatment of neurological diseases by interferon antagonists

ABSTRACT

The present invention relates to a process for ameliorating or preventing neurological diseases that are caused, in part, by an increased and/or abnormal responsivity to interferon. Specifically, the invention provides a method for treating subjects suffering from or at risk for such diseases by the administration of a pharmacological preparation that antagonizes interferons&#39; action.

[0001] This application is a continuation of U.S. patent applicationSer. No. 08/502,519, filed Jul. 14, 1995.

1. FIELD OF THE INVENTION

[0002] The present invention relates to a process for ameliorating orpreventing neurological diseases that are caused, in part, by anincreased and/or abnormal responsivity to interferon. Down Syndrome (DS)and Alzheimer's Disease (AD) are examples of such diseases.Specifically, the invention provides a method for treating subjectssuffering from or at risk for such diseases by the administration of apharmacological preparation that antagonizes interferons' action.

2. BACKGROUND OF THE INVENTION

[0003] 2.1. The Molecular Biology of Interferons and InterferonReceptors

[0004] Interferons are proteins that alter and regulate thetranscription of genes within a cell by binding to interferon receptorson the regulated cell's surface and thus prevent viral replicationwithin the cells. There are five types of interferons (IFN), which aredesignated α (formerly α₁), ω (formerly α₂), β, γ and τ. Mature humaninterferons are between 165 and 172 amino acids in length. In humansIFN-α and IFN-ω are encoded by multiple, closely related non-allelicgenes. Additionally, there are pseudo-genes of IFN-α and IFN-ω. Bycontrast, IFN-β and IFN-γ are encoded by unique genes.

[0005] The interferons can be grouped into two types. IFN-γ is the soletype II interferon; all others are type I interferons. Type I and typeII interferons differ in gene structure (type II interferon genes havethree exons, type I one), chromosome location (in humans, type II islocated on chromosome-12; the type I interferon genes are linked and onchromosome-9), and the types of tissues where they are produced (type Iinterferons are synthesized ubiquitously, type II by lymphocytes). TypeI interferons competitively inhibit each others binding to cellularreceptors, while type II interferon has a distinct receptor. Reviewed bySen, G. C. & Lengyel, P., 1992, J. Biol. Chem. 267:5017-5020.

[0006] Although all type I interferons compete for binding to a commonreceptor or receptors, the effects of different type I interferons canbe different. Pontzer, C. H., 1994, J. Interfer. Res. 14:133-41.Additionally, there appears to be several kinds of type I interferonreceptor. For example, there is evidence that the type I interferonreceptors of different cell types are different. Benoit, P., 1993, J.Immunol. 150:707. The number of genes encoding the type I interferonreceptors is unknown: however, the genes appear to be linked to eachother and to at least one gene encoding an IFN-γ receptor component aswell. In humans, chromosome region 21q21.1-21.31 encodes all the genesneeded for the receptor for type I interferon (Raziuddin, A., 1984,Proc. Natl. Acad. Sci. 81:5504-08; Soh, J., 1993, Proc. Natl. Acad. Sci.90:8737-41; Soh, J., 1994, J. Biol. Chem. 269:18102-10) and at least oneessential component of the type II interferon receptor (Jung, V., 1990,J. Biol. Chem. 265:1827-30).

[0007] 2.2. The Biology of Interferon Action and Down Syndrome

[0008] The binding of interferons to their receptor, leads to a cascadepost-translational modification to other proteins which are thentransported to the nucleus where they regulate the transcription ofgenes by binding to specific nucleic acid sequences. The nucleic acidsequence which is characteristic of genes responsive to type Iinterferons is designated the Interferon Sensitive Response Element(ISRE). Reviewed Tanaka, T. & Taniguchi, T., 1992, Adv. Immunol. 52:263.Type-I interferons are synthesized in response to viral infection,except for IFN-τ which is constitutively produced in the placenta; TypeII interferons are synthesized in response to antigen stimulation.

[0009] Interferons alter the rates of synthesis and the steady statelevels of many cellular proteins. An overall effect of interferon isusually an inhibition of cellular proliferation.

[0010] The possibility that cells from subjects having Down Syndrome mayhave abnormal responsivity to interferon was introduced by the discoverythat a gene encoding an interferon inducible protein, which wassubsequently identified as the type I interferon receptor, was locatedon chromosome-21. Tan Y. H. et al., 1974, J. Exp. Med. 137:317-330. Thisobservation prompted comparisons of the response of diploid andtrisomy-21 aneuploid cultured cells to interferon added to the culturemedium. These studies have consistently shown an increased responsivityof trisomy-21 cells to interferon. Tan, Y. H., et al., 1974, Science186:61-63; Maroun, L. E., 1979, J. Biochem. 179:221; Weil, J., et al.,1983, Hum. Genetics 65:108-111; reviewed Epstein, C. J., & Epstein, L.B., 8 LYMPHOKINES pp277-301 (Academic Press, NY, 1983); Epstein, C. J.et al., 1987, ONCOLOGY AND IMMUNOLOGY OF DOWN SYNDROME (Alan R. Liss,1987). The publications of these studies have been accompanied byspeculative conjectures that the altered responsivity to interferonplayed a role in the pathogenesis of lesions of Down Syndrome. See,Maroun, L. E., 1980, J. Theoret. Biol. 86:603-606.

[0011] 2.3. Down Syndrome and Animal Models of It

[0012] An animal model of Down Syndrome has been constructed by use ofthe knowledge that human chromosome-21 is syntenic to mousechromosome-16, i.e., that many of the genes present on each are homologsof each other. Mice having specified trisomies can be bred by use ofparental mice having “Robertsonian” chromosomes, i.e., chromosomes thatare essentially the centromeric fusion of two different murinechromosomes. A variety of such Robertsonian chromosomes have beenidentified, including at least two involving chromosome-16 and a seconddifferent chromosome: Rb(16.17) and Rb(6.16). Mice homozygous for anyRobertsonian or combination of independent Robertsonian chromosomes areeuploid and fertile. The intercross (F₁) between an Rb(16.17) and anRb(6.16) mouse is also fully diploid at each genetic locus, althougherrors in meiosis may cause reduced fertility. Note that in such an F₁both the maternal and paternal chromosome-16 are a part of aRobertsonian chromosome.

[0013] Because of meiotic errors the outcross between a mouse havingboth two different Robertsonian chromosome-16's and a non-Robertsonianmouse gives rise to a trisomy-16 conceptus in between 15% and 20% ofcases. Gearhart, J. D., et al., 1986, Brain Res. Bull. 16:789-801;Gropp, A., et al., 1975, Cytogenet. Cell Genet. 14:42-62. The murinetrisomy-16 fetuses develop to term but do not live beyond birth by morethan a few hours.

[0014] Examination of the fetal trisomy-16 and the post-partum humantrisomy-21 reveals a number of analogous or parallel lesions. For thisreason, the murine trisomy-16 construct-is considered to be an animalmodel of Down Syndrome. Epstein, C. J., THE METABOLIC BASIS OF INHERITEDDISEASE, 6TH ED. pp291-326 (McGraw-Hill, NY, 1989); Epstein, C. J., etal., 1985, Ann, N. Y. Acad. Sci. 450:157-168. Because a murinetrisomy-16 fetus is not viable post partum, the opportunity to study theneurological pathology of the model has been limited. However, it isclear that in both human trisomy-21 and murine trisomy-16 there is anoverall reduction in fetal size and particularly in the development ofthe fetal brain. Epstein, C. J., THE CONSEQUENCES OF CHROMOSOMEIMBALANCE: PRINCIPLES, MECHANISMS AND MODELS (Cambridge UniversityPress, NY, 1986). Further insights into the effects of murine trisomy-16have been obtained by the formation of Ts16++2N chimeras (Gearhart, J.D., et al., 1986, Brain Res. Bulletin 16:815-24) and by transplantationof fetal-derived Ts16 tissue into a 2N host (Holtzman, D. M., et al.,1992, Proc. Natl. Acad. Sci. 89:1383-87; Holtzman, D. M., et al., DOWNSYNDROME AND ALZHEIMER DISEASE, pp227-44 (Wiley-Liss, NY, 1992).

[0015] 2.4. Alzheimer'S Disease and Amyloid Precursor Protein

[0016] Alzheimer's Disease is a progressive dementia which ischaracterized by the precipitation of a peptide, termed an Aβ peptide,of about 40 amino acids within the brain and within the walls of bloodvessels in the brain. The Aβ peptide is derived from the processing of alarger cell surface protein called the β Amyloid Precursor Protein(βAPP). Production of the Aβ peptide is not per se pathological. Thefunctions of both the Aβ peptide or βAPP are unknown.

[0017] Several lines of evidence indicate that the deposition of the Aβpeptide is not merely correlative but rather causative of Alzheimer'sDisease. The gene encoding βAPP is located on chromosome-21 and, asnoted above, subjects having Down Syndrome develop Alzheimer's Disease.More directly, kinship groups have been identified among the many causesof familial Alzheimer's Disease in which the inheritance of the Diseaseis linked to the inheritance of a gene encoding a mutated βAPP, moreoverthe mutation is within the Aβ peptide itself. Reviewed Selkoe, D. J.,1994, Ann. Rev. Neurosci. 17:489-517. Transgenic mice, having multiplecopies of such a mutant βAPP gene, operatively linked to a strong,neuronal and glial cell specific promoter, develop the anatomicallesions of Alzheimer's Disease at about 6-9 months of age. Games, D., etal., 1995, Nature 373:523.

[0018] There is a relationship between Down Syndrome and Alzheimer'sDisease. The gene encoding the βAPP is found on chromosome-21. Patientswith Down Syndrome are at increased risk of developing Alzheimer'sDisease, most often by about the fifth decade of life although cases ofearlier development have been reported. Mann, D. M. A., et al., 1990,Acta Neuropathol. 80:318-27.

3. SUMMARY OF THE INVENTION

[0019] The present invention is based, in part, on the recognition thatin certain pathologic processes that result in mental impairment, thehost is rendered abnormally and/or aberrantly sensitive to the effectsof interferon so that the effects of interferon become an immediate anddirect cause of the pathology. Such processes include, in humans,trisomy of chromosome-21 or the portion of the chromosome-21 thatencodes the receptor for type I interferon and at least one component ofthe receptor for IFN-γ, which is the genetic abnormality associated withDown Syndrome; and also include Alzheimer's Disease.

[0020] The present invention provides a method of ameliorating thepathologic effects of interferon by administering to a subject, in theabove-noted circumstances, an antagonist of interferon. Embodiments ofthe invention include the administration of antagonists, alone or incombination, that are antagonists of Type I interferon, Type IIinterferon (IFN-γ), and placental interferon (IFN-τ).

4. DESCRIPTION OF THE FIGURES

[0021]FIG. 1A-C. The lengths of Trisomy 16 fetuses plotted as a functionof the average length of normal littermates.

[0022]FIG. 1A, Uninjected controls; FIG. 1B, non-specific IgG (ns-IgG)injected controls; FIG. 1C, anti-IFN injected fetuses. Ananalysis-of-covariance was performed to compare the groups on lengthwhile adjusting for average normal littermate length. The lengths of theanti-IFN treated group were significantly greater than those of thens-IgG injected controls (p=0.0112) and those of the uninjected controls(p=0.0037). The dotted lines in each figure encompass the 95% confidencelimits.

[0023]FIG. 2A-B. Morphometric analysis of the development in normal,Trisomy 16 treated and Trisomy 16 sham treated fetuses. FIG. 2A, averageeye opening of 17 to 23 mm trisomy 16 fetuses; FIG. 2B, average backcurvature scores of trisomy 16 fetuses greater than 20 mm in length.Columns: (A) Uninjected; (B) non-specific IgG injected; (C) anti-IFNinjected; (D) euploid. The mean ± standard error is presented.

5. DETAILED DESCRIPTION OF THE INVENTION

[0024] 5.1. Selection of Subjects

[0025] The present invention concerns the administration of interferonantagonists to subjects in order to ameliorate the neurological anddevelopmental abnormalities in the subject due to the action ofinterferon. A particular group of subjects at risk are subjects having atrisomy of the portion of the chromosome region, designated in humans21q21.1-21.31, that encodes for interferon receptors. This group has theclinical diagnosis of Down Syndrome. Grete, N., 1993, Eur. J. Hum.Genetics 1:51-63; Sinet, P. M., 1994, Biomed. & Pharmacol. 48:247-252.The homologous chromosome in mice is chromosome-16.

[0026] Diagnosis of Down Syndrome can be made by any method known to themedical arts. Typically, for diagnosis in utero, amniocentesis can beperformed at about 14 weeks of gestational age and chorionic villussampling (biopsy) can be performed between 9 and 12 weeks of gestationalage. Down Syndrome in children and adults is diagnosed from karyotypesof peripheral blood cells. Cells from either type of sample are culturedand cytogenetic examination can be performed by methods well understoodby those skilled in the art.

[0027] As noted above, subjects having Down Syndrome are at increasedrisk to develop Alzheimer's Disease. A further group of subjects thatwould benefit from the invention consist of subjects having thediagnosis of probable Alzheimer's Disease or who are at increased riskof developing Alzheimer's Disease from causes other than Down Syndrome.The diagnosis of probable Alzheimer's Disease is made by clinicalcriteria (McKhann, G., 1984, Neurology 34:939; DIAGNOSTIC ANDSTATISTICAL MANUAL OF MENTAL DISORDERS IV, American PsychologicalAssociation, Washington, D.C.). Persons having a familial predispositionto Alzheimer's Disease are also suitable subjects for the presentinvention.

[0028] 5.2. The Selection of Antagonists

[0029] The antagonist of the invention can be any antagonist that can beadministered to the subject in an amount effective to prevent thedeleterious action of the interferon on the central nervous system.

[0030] The effective amount of antagonists that act by binding to andblocking interferon proteins in the blood can be determined by assayingthe concentration of bioavailable interferon in the subjects blood. Aneffective dose of antagonist is a dose that is sufficient to reduce thelevel of bioavailable interferon by between at least three to five fold,more preferably by about ten fold and most preferably by about twentyfive fold below the normal levels of interferon.

[0031] The assay of bioavailable interferon is performed by adding asample of the subjects blood to a culture of an interferon sensitivecell line which is then infected with a test virus, typically VesicularStomatitis Virus (VSV), and the number of viral plaques is determined orthe cytotoxic effects of the VSV infection is otherwise quantitated.Bioavailable interferon blocks productive viral infection. The level ofbioavailable interferon is calculated by comparing various dilutions ofthe test sample with a titration of a standard sample of interferon.Such assays are rountine in the art. See, e.g., Hahn, T., et al., 1980,in INTERFERON: PROPERTIES AND CLINICAL USES, ed. by A. Khan, N. O. Hilland G. L. Dorn, (Leland Fikes Foundation Press, Dallas, Tex.);Armstrong, J. A., 1971, Applied Microbiology 21:723-725; Havell, E. A. &Vilcek, J. 1972, Anti-microbial Agents and Chemotherapy 2:476-484.

[0032] In one embodiment of the invention the antagonist is a monoclonalanti-interferon antibody or fragment thereof. The production of suchantibodies is well known in the art. The production of anti-IFN-αmonoclonal antibodies that block interferon activity is taught by U.S.Pat. No. 4,973,556 to Bove et al. The production of blocking monoclonalantibodies to IFN-γ is taught by U.S. Pat. No. 4,948,738 to Banchereau.The structure of human trophoblastic interferon (IFN-τ) has beenrecently disclosed (Whaley, A. E., 1994, J. Biol. Chem. 269:10864-8).Monoclonal antibodies and other antagonists to this interferon can beproduced using methods well known to those skilled in the art.

[0033] In a preferred embodiment, the antibody is a “chimeric” antibody,i.e., an antibody having a variable region from one species and aconstant region from another species. Most typically chimeric antibodiesfor use in humans have constant regions of human origin. In analternative preferred embodiment, the antibody is a “grafted” antibody,i.e., an antibody having complementarity determining regions from onespecies and a constant region and a framework region of the variableregion from a second species. A grafted antibody in which the secondspecies is human is termed a “humanized” antibody. Methods of makingchimeric antibodies suitable for pharmaceutical use are disclosed inpatent publication W092/16553 by Le, J. (Oct. 1, 1992). “Grafted”antibodies and “humanized” antibodies are described in U.S. Pat. No.5,225,539 to Winter and patent publications W091/09967 and W092/11383 byAdair, J. R. et al. Suitable antagonists, smaller than an antibodymolecule, can be derived from anti-interferon monoclonal antibodies bytechniques well known in the art. See, e.g., U.S. Pat. No. 5,091,513 toHuston and U.S. Pat. No. 5,260,203 to Ladner. As used herein the term“antibody antagonists” includes natural polyclonal and monoclonalantibodies, chimeric and grafted antibodies, and enzymatically andrecombinantly produced interferon binding fragments of each type ofantibody.

[0034] In an alternative embodiment the antagonist can be arecombinantly produced protein that comprises the interferon bindingportion of an interferon receptor. The production of soluble interferonreceptors by baculovirus transduced cells is described in Fountoulakiset al., 1991, Eur. J. Biochem. 198:441-450. Alternatively the antagonistcan be a fusion protein that contains an interferon binding domain of aninterferon receptor.

[0035] In alternative embodiments, the antagonist can be an antibody toan interferon receptor, a soluble interferon receptor, receptorfragment, or a peptide that is derived from an interferon that occupiesthe receptor binding site but does not activate the receptor. Such anIFN-γ peptide antagonist is disclosed by Jarpe, M. A. et al., 1993, J.Interferon Res. 13:99-103.

[0036] When the subject is a fetus, or an infant less than 6 weeks ofage, the blood brain barrier is not fully formed. In these circumstancesantibodies and other proteins that block the interferon receptor candirectly reach the central nervous system. When the subject has anintact blood brain barrier, the preferred embodiment of the inventionemploys antibodies and proteins that block interferon by binding theinterferon directly, rather than those that act at the interferonreceptor.

[0037] Alternatively, increased CNS entry of antibody antagonists can beobtained by chemical modification of the antagonist. Such modificationsinclude cationization, Pardridge, W., 1991, “Peptide Drug Delivery tothe Brain”, and glycation, Poduslo, J. F., & Curran, G. L., 1994,Molecular Brain Research 23:157.

[0038] The interferon antagonist can be a mixture of antagonists thatare specific for the various different types of interferon. When onetype of interferon predominates, the antagonist can be an antagonist foronly the predominate type of interferon that is present. For example,when the subject is a fetus, the antagonist can be a INF-τ specificantagonist.

[0039] When the subject is a fetus, then the antagonist can beadministered by a transplacental route, e.g., antibody that istransported across the placenta. The human isotypes IgG1, IgG3 and IgG4are suitable for transplacental administration.

[0040] 5.3. Selection of Dose and Timing of Administration

[0041] The amount of an antibody antagonist administered is between 1and 100 mg/kg. The preferred route of administration of an antibodyantagonist is intravenous administration to infant and adult subjects.The preferred route of administration to fetal subjects is byintravenous administration to the mother followed by transplacentaltransport. Alternatively antibody antagonists can be administered byintramuscular and subcutaneous routes. When an antagonist is deliveredtransplacentally, the calculation of the dose is based on the maternalweight.

[0042] The antagonist is administered to subjects having Down Syndromepreferably at the time when the central nervous system is developingmost rapidly. The preferred period of administration is from agestational age of 24 weeks onwards until a post natal age of about 2years. Even though some proliferation of neurons takes place duringweeks 8-18, it is not critical that an antagonist be administered to ahuman subject prior to week 20-24 of gestational age because thesynaptic connections between the neurons are not formed until week 20.Brandt, I., 1981, J. Perinat. Med. 9:3. The administration of theantagonist to subjects having Alzheimer's Disease should commence at thetime that the diagnosis of probable Alzheimer's Disease is first madeand continue there after. In middle age, subjects having Down Syndromedevelop a dementia having an anatomical pathology which is identical toAlzheimer's Disease (Mann, D. M. A., 1988, Mech. Aging and Develop.43:99-136). Thus, the administration of the antagonist to Down Syndromepatients can be continued throughout the life of the patient, as DownSyndrome patients are at risk for Alzheimer's Disease ab initia.

[0043] The frequency of administration is determined by the circulationtime of the antagonist, which can be determined by direct measurement bymethods well known to those skilled in the art.

[0044] In an alternative embodiment of the invention, the administrationof interferon antagonists is replaced by the extracorporeal treatmentsof the subject's blood to remove circulating interferon, such as isdescribed in U.S. Pat. No. 4,605,394.

[0045] 5.4. A Model Embodiment of the Invention

[0046] The invention is exemplified and its operability is demonstratedby the experiments that are presented in Example 1 below. Briefly,normal female mice were crossed with double heterozygous males havingRb(6.16) and Rb(16.17) chromosomes. The females were injected with amixture of rat monoclonal anti-IFN-γ (1500 neutralizing units) andrabbit polyclonal anti-IFN-α/β (1362 neutralizing units)interperitonally (i.p.) on days 8, 10, 12 and 14 of pregnancy. On day 17the embryos were biopsied for cytogenetic classification, sacrificed andfour gross parameters were measured and compared to the geneticallynormal littermates in order to assess relative development. Controlgroups consisted of untreated females and sham treated females whichwere given normal rabbit and rat serum γglobulin injections.

[0047] The four measured parameters were overall (crown-rump) length ofthe fetus, shape of the back (normally concave at birth), eye-closing(the eyes normally close shortly before birth) and fetal weight. Theresults of the comparison of each of the parameters from 17 untreated,16 sham treated and 18 treated controls showed a statisticallysignificant reduction in the growth retardation/maturation of thetreated trisomy-16 fetal mice compared to their euploid littermates.

[0048] The fetuses from anti-IFN treated mothers had a mean weightdecrease of −10.92% compared to a −21.47% decrease for the uninjectedgroup (p=0.079) and a −30.46% decrease for the ns-IgG injected group(p=0.0003) relative to diploid littermates. The uninjected and ns-IgGinjected control groups were not statistically different from each other(p=0.174).

6. EXAMPLE TREATMENT of Murine Trisomy-16 by a Interferon Antagonist

[0049] 6.1. Materials and Methods

[0050] Animals and Mating. 6:16 Robertsonian translocation male(Rb[6.16]24Lub) and 17:16 Robertsonian translocation female(Rb[16.17]7Bnr) homozygotes were purchased from Jackson Laboratories,Bar Harbor, Me. Mature (54 day) male offspring of these homozygotes(double heterozygotes) were mated to 8-10 wk old euploid, nulliparous,C3H/HeJ females (Jackson Laboratories). Surgery was performed on day 17or 18 to yield fetuses at the 17-25 mm stage (Theiler, K. (1972) In: TheHouse Mouse, Springer, Berlin, Heidelberg, New York). The last threedays of gestation are when the morphologic characteristics (eye closure,back curvature and accelerated growth) can be quantified.

[0051] Injections. Intraperitoneal (IP) injections (0.25 cc) were begunon post-coitus day 8 (implantation occurs on day 5.5). Injections weregiven every 48 hours for a total of four injections per animal.

[0052] Rabbit polyclonal anti-mouse α/β IFN purified IgG (970neutralizing units/mg of protein, cat. #25301), and rat monoclonal IgG1anti-mouse γ IFN, (7,200 neutralizing units/mg, cat. 25001) wereobtained from Lee Biomolecular Research Incorporated, San Diego, Calif.The anti-IFNs (supplied lyophilized from saline) were dissolved insterile water-for-injection (Investage) at a concentration that woulddeliver 1500 neutralizing units of anti-γ and 1362 neutralizing units ofanti-α/β IgG per injection. The expectation was that the IgG would reachthe developing fetus through active IgG placental transfer(Guzman-Enriques, L., et al., 1990, J. Rheumatol., 17:52-56). Controlinjections delivered the same mg quantities of rat (Pierce cat. #31233X)and rabbit (Pierce cat. #31207X) non-specific IgGs in an equivalentvolume of sterile saline-for-injection (Abbott). A second control groupconsisted of uninjected mothers which were left undisturbed.

[0053] Fetus Processing. Fetuses, obtained by hysterectomy of micesacrificed by cervical dislocation, were photographed, measured andfixed whole in Bouins fixative (Luna, L. G. (1968) In: Manual ofHistologic Staining Methods of the Armed Forces Institute of Pathology,(3rd edition). The Blakiston Division, McGraw-Hill Book Company, NewYork). Prior to fixation, limb tissue was obtained and minced to providefibroblast cultures for karyotyping. The fetal fibroblasts from theminced tissue were grown at 37° C. in EAGLE's Minimum Essential Mediacontaining 20% fetal bovine serum, 2 mM glutamine, 100 units/ml ofpenicillin, and 100 μg/ml of streptomycin. After five days in culture,colchicine (Sigma) was added to level of 1 μg/ml. One hour later, cellswere collected, swelled in 25% media, and fixed in fresh methanol:acetic acid (3:1). Crown-to-rump length was measured immediately afterthe fetus was obtained by measuring the vertex-to-rump distance (withoutpressure on the fetus) while the fetus was floating in serum-freeMinimum Essential Media. Except where otherwise noted, all statisticalanalyses were done using a two-tailed student's T-test.

[0054] 6.2. Results and Discussion

[0055] Mice pregnant with trisomy 16 conceptuses were obtained by themating of euploid nulliparous C3H/HeJ females with doubly heterozygousmales. The males were also functionally euploid (i.e., they have a totalof 40 chromosome arms) but they carried two Robertsonian translocationchromosomes (6.16 and 17.16), each with one chromosome #16 arm. Themeiotic misdistribution of these translocation chromosomes results in ahigh frequency of trisomy 16 fetuses carrying a maternal acrocentricchromosome 16 and both paternal translocation pseudometacentricchromosomes. This genetic system has been described in detail (Gropp,A., et al., 1975, Cytogenet. cell Genet. 14:42-62; Gearhart, J. D., etal., 1986, Brain Res. Bull. 16:789-801). Anti-IFN treated mothersreceived four IP injections of a cocktail of anti-α, β and γ IFNimmunoglobulins. One control group of mothers was left unhandled and onewas given comparable injections of non-specific IgG.

[0056] Mechanisms for the transfer of the IgG from mother-to-fetus andneonate vary widely from species to species. Generally, some combinationof passive and active transport is involved; sequentially utilizing theyolk sac and placenta prior to birth, and the intestine postnatally. Inthe mouse system, maternal antibodies can initially be found in thefluid filling the blastocyst cavity (Brambell, F. W. R., 1966, TheLancet 7473). This may be due simply to passive diffusion, as this fluidgenerally resembles dilute maternal blood plasma. Shortly thereafteractive transport of IgG class immunoglobulins via Fc receptors becomesprimarily the function of the yolk sac. This function is later sharedbut, in rodents, never dominated by Fc mediated transfer of IgG acrossthe placenta (Roberts, D. M. et al., 1990, J. Cell Biol. 111:1867-1876).In the experiments presented here, mice were injected after day 5.5because of the possibility that trophoblast interferon may play animportant role at implantation (Roberts, R. M., 1991 BioEssays13:121-126). In the mouse, injected polyclonal rabbit IgG has anexpected half-life of approximately 5 days (Spiegelberg, H. L. & W. O.Weigle, 1965, J. Exp. Med. 121:323-337).

[0057] A total of 68 late stage fetuses with abnormal morphology wereobtained from among 440 offspring of 143 doubly heterozygousmale×C3H/HeJ female matings. Only fetuses that were both successfullykaryotyped and from litters where euploid fetuses averaged greater than17 mm in length (crown-to-rump [CRL]) are included in TABLE 1 and in allgraphs. Fifty-one of a total of 68 trisomies met these criteria. In allcases, the return-toward-normal values are seen with or without theinclusion of unkaryotyped fetuses. For comparison, p values calculatedwith the unkaryotyped fetuses included are provided in brackets next tothose calculated using only successfully karyotyped fetuses.

[0058] Growth Retardation. The growth retardation seen in the trisomy 16fetus is quite variable. Nonetheless, the trisomic fetuses from theanti-IFN treated mothers showed a significant return-toward-normalgrowth when CRL length is plotted against the average length of theeuploid littermates (FIG. 1). This analysis suggests that unlike theerratic growth of their counterparts from untreated mothers, the trisomy16 fetuses from anti-IFN treated mothers were nearly keeping pace withthe growth of their euploid littermates.

[0059] On average the trisomic fetuses from anti-IFN treated mothersshowed a 5.6% decrease in length compared to a 15.28% decrease for thefetuses from non-specific IgG injected mothers (p=0.014 [0.0009]) and a14.59% decrease for the fetuses from uninjected mothers (p=0.015[0.010]). The two control groups were not statistically different fromeach other (p=0.879 [0.759]). The improvement in growth was seenconsistently against both control groups and in all the fetus sizegroups (17-20 mm, 20-23 mm, >23 mm, Table 1).

[0060] A similar return-toward-normal growth was observed when thedecrease in trisomy 16 fetal weights were analyzed. The fetuses fromanti-IFN treated mothers had a mean weight decrease of −10.92% comparedto a −21.47% decrease for the uninjected group (p=0.079 [0.095], NS) anda −30.46% decrease for the ns-IgG injected group (p=0.0003 [0.0026]).The two control groups were not statistically different from each other(p=0.174 [0.33]).

[0061] There were no detectable effects of the non-specific IgG oranti-IFN injections on the euploid fetuses. Growth of each trisomicfetus was measured against its normal littermates to avoid errors due toa missed estimate of gestational age. In these matings, the mean normallittermate length (MNLL) measured 17.17 mm CRL at gestational day 16.5,19.39 mm CRL at day 17.5 and 23.94 mm CRL at day 18.5 (plug date=day 0.5[Kaufman'92]). There was no significant difference between the MNLL ofthe uninjected control group (gestational day) 18.5 (MNLL=23.944 [N=18,p=0.419])or the IgG injected control group (MNLL=23.75 [N=6, p=0.706]),and the anti-IFN treated group (MNLL=23.333 [N=24]). There was also nosignificant difference between the MNLL of the two control groups(p=0.826).

[0062] Eye Opening. Eye opening comparisons (FIG. 2A) were limited tofetuses from litters 17 mm to 23 mm in length. Prior to this stage allfetuses have open eyes. The eyes of fetuses from litters measuring16.9-22.6 mm CRL obtained from anti-IFN treated mothers (N=13, mean=0.21mm) had made significantly more progress toward closure than the eyes ofcomparably staged fetuses from untreated (N=11, mean=0.42 mm, p=0.19[0.010]) and non-specific IgG injected mothers (N=11, mean=0.40 mm,p=0.026 [0.046]). There was no significant difference in the eyeopenings of the uninjected and non-specific IgG injected control groups(p=0.746 [0.300]). Progress toward eye closure may be an all or nothingevent. Thus, it may be equally significant that 7 of the 13 fetuses(54%) from anti-IFN treated mothers had eye openings that averaged lessthan 0.2 mm compared to 2 of 11 (18%) of those from untreated mothersand 2 of 11 (18%) of the comparable fetuses from non-specific IgGtreated mothers.

[0063] There have been numerous mutations detected in the mouse thatlead to open eyelids (Teramoto, S, et al., 1988, Exp. Anim.,37:455-462). Most of these mutations show complete penetrance. However,some affect each eye variably and at least one phenotype can be reversedby a single maternal injection of steroids (Watney, M. J., & J. R.Miller, 1964, Nature 202:1029-1031). In addition, phenocopies of theseutants can be induced by common teratogens (Juriloff, D. M., et al.,1982 Can. J. Genet. Cytol., 25:246-254). The eyelid is lined with anactive zone of cell growth (Kaufman, M. H., 1992, In: The Atlas of MouseDevelopment. Academic Press, Harcourt Brace Jovanovich, San Diego,Calif.), and these data indicate that the effect of the anti-IFNantibodies is to block cell growth inhibition of the interferonsuper-sensitive trisomy 16 cells lining the eyelids.

[0064] Back Curvature. One of the most striking effects of the aternalanti-IFN treatment was the return-toward-normal of the curvature of thetrisomy 16 fetus back which is frequently rounded at later stages wherea concave curvature is expected. Back curvature comparisons (FIG. 2B)are restricted to fetuses from litters greater than 20 mm in lengthbecause both euploid and trisomic fetuses are expected to have roundedbacks prior to the 20 mm stage (Theiler, K., 1972, In: The House Mouse,Springer, Berlin, Heidelberg, New York). Back curvature was assessed bya double-blind study in which three individuals scored a rounded back asa −1, a flat back as a 0 and a convex (normal) back as a +1. There wasgood agreement between the scores of the three individuals (correlationsranged from 0.80 to 0.92). The mean of the three evaluations was usedfor comparisons.

[0065] There was no significant difference in the back curvature scoresof the trisomic fetuses from uninjected and non-specific IgG injectedcontrol mothers (p=0.8236 [0.3424]). The trisomic fetuses from anti-IFNtreated mothers (N=10, mean=+0.66) showed a significantreturn-toward-normal back curvature when compared to fetuses fromuntreated mothers (N=9, mean =−0.18, p=0.009 [0.009]) and the comparablefetuses from non-specific IgG treated animals (N=11, mean=−0.27, p=0.008[0.003]).

[0066] One hundred fifty fetuses whose eyes, back, and length, appearednormal were, also karyotyped (75 control and 75 anti-IFN treated). A 24mm fetus was one of two fetuses discovered to be trisomy in this screen.A second fetus (10 mm CRL) was also found in a litter from an anti-IFNtreated mother and was essentially indistinguishable from its euploidlittermates. LEGEND, TABLE 1: Compilation of data on karyotyped trisomy16 fetuses.

[0067] (A) Mean length of normal littermates (mm, CRL); (B) Length oftrisomic fetus (mm, CRL); (C) Change in trisomic fetus length relativeto its normal littermates (%); (D) Average weight of normal littermates(gm); (E) Weight of trisomy fetus (gms); (F) Opening of the eyes (mm);(G) Average back curvature scores of three individuals, +1=normalconcave, 0=flat, −1=rounded.

[0068] 7. Example Construction of a Recombinant Interferon AntagonistComprising Human Interferon α/β and γ Receptor Domains

[0069] A gene encoding a fusion protein is constructed using aGlutamine-S-transferase containing expression plasmid pAcGHLT-B(Pharmingen). The interferon binding domain of the human α/β interferonreceptor is obtained by Nco I endonuclease digestion of plasmid p23,available from deposit No. ATCC 65007, and isolation of the 1177 bpfragment. This fragment is inserted into the Nco I site of pAcGHLT-B toyield pAcGST-23. The interferon binding domain of the human γ interferonreceptor is obtained by Dsa I and Nsp I endonuclease digestion of theplasmid pUCLGRIF16, available from deposit No. ATCC 59873, and isolationof the 603 bp fragment. A Pst I-Sma I digest of pAcGST-23 is used toremove a portion of the multiple cloning site located 3′ of the geneencoding the α/β interferon receptor domain and the Dsa I/Nsp I fragmentof pUCLGRIF16 is inserted to yield pAcGST-23-γr. The translation productof the resultant construct, GST-α/β-γ, contains the following domains:GST-thrombin protease site-15 amino acid leader-α/β interferon receptordomain-6 amino acid spacer-γ interferon receptor domain.

[0070] A recombinant baculovirus is constructed containing thepAcGST-23-γr operably linked to the polyhedrin promoter, suitable hostcells are infected and the resultant fusion protein isolated by ananti-GST affinity absorption techniques well known in the field. See,e.g., U.S. Pat. Nos. 4,745,071 and 4,879,236 to Smith et al. Theisolated fusion protein is hydrolyzed with thrombin to yield therecombinant α/β-γ receptor.

[0071] The present invention is not to be limited in scope by thespecific embodiments described which were intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims. All references are hereby incorporated by reference in theirentirety. TABLE 1 (A) (B) (C) (D) (E) (F) (G) Ln NLM Ln Tri % ↓ Wt NLMWt Tri eyes Back curv. (mm)[N] (mm)[N] (mean ± sem) (gm)[N] (gm)[N] (mm)± sem [N] (euploid = +1) Uninjected 17-20 mm 18.32[5] 17.29[8]  −4.12 ±1.7 0.60[5] 0.488[8] 0.490 ± 0.03[8] −0.207 62 fetuses (17 trisomic),20-23 mm 21.26[3] 16.83[3] −20.67 ± 5.5 0.84[3] 0.591[3] 0.237 ± 0.12[3]+0.333 5 resorption sites, 13 litters  >23 mm 24.63[5] 18.40[6] −25.50 ±4.3 1.21[5] 0.749[6] 0.200 ± 0.06[6] −0.443 (mean litter size: 4.8)ns-IgG 17-20 mm 18.30[4] 16.20[5] −11.20 ± 4.0 0.614[4] 9.463[5] 0.530 ±0.03[5] −0.266 60 fetuses (16 trisomic), 20-23 mm 20.96[4] 18.30[6]−12.60 ± 2.2 0.796[4] 0.526[6] 0.283 ± 0.04[6] −0.220 2 resorptionsites, 13 litters  >23 mm 25.90[5] 19.80[5] −22.60 ± 9.2 1.460[5]0.911[5] 0.127 ± 0.04[5] −0.334 (mean litter size: 4.6) Anti-IFN 17-20mm 18.92[5] 18.66[8]  −2.80 ± 2.4 0.50[5] 0.657[8] 0.230 ± 0.06[8]−0.165 70 fetuses (18 trisomic), 20-23 mm 20.57[4] 19.14[5]  −7.10 ± 3.20.78[4] 0.643[5] 0.176 ± 0.06[5] +0.530 4 resorption sites, 13 litters >23 mm 24.59[4] 22.38[5]  −8.40 ± 1.7 1.24[4] 1.060[5] 0.035 ± 0.03[5]+0.800 (mean litter size: 5.4)

What is claimed is:
 1. A method of ameliorating the pathological effectsof a trisomy of an autosomal chromosome or portion thereof whichcomprises administering an amount of an interferon antagonist to amammal having a trisomy that renders the cells of the mammalhypersensitive to interferon, said amount being effective to amelioratethe pathological effects of the trisomy.
 2. The method of claim 1wherein the mammal is a human and the autosomal chromosome ischromosome-21.
 3. The method of claim 2 wherein the antagonist blocksproduction of interferon.
 4. A method of treating dementia whichcomprises administering to a human subject an amount of aninterferon-binding interferon antagonist that is effective to reduce thelevel of bioavailable interferon in the subject's blood to at most onethird of a normal level of bioavailable interferon, wherein the humansubject has a dementia.
 5. The method of claim 4 wherein the dementia isa type of dementia that is associated with an accumulation of amyloid inthe central nervous system of the subject.
 6. The method of claim 4wherein the interferon antagonist is an antibody antagonist.
 7. Themethod of claim 4 wherein the interferon antagonist comprises aninterferon receptor interferon-binding domain.
 8. The method of claim 4wherein the antagonist blocks production of interferon.
 9. A method ofpreventing a disease of the central nervous system of a mammaliansubject which comprises administering to a mammalian subject an amountof an interferon-binding interferon antagonist that is effective toreduce the level of bioavailable interferon in the subject's blood to atmost one third of a normal level of bioavailable interferon, wherein thesubject is at increased risk to accumulate amyloid in the centralnervous system.
 10. The method of claim 9 wherein the subject is a humanwho is at increased risk of developing Alzheimer's Disease.
 11. Themethod of claim 10 wherein the interferon antagonist is an antibodyantagonist.
 12. The method of claim 10 wherein the interferon antagonistcomprises an interferon receptor interferon-binding domain of aninterferon receptor.
 13. The method of claim 9 wherein the antagonistblocks production of interferon.
 14. A method of treating dementia whichcomprises administering to a human subject an amount of an interferonantagonist which blocks the action of an interferon receptor that iseffective to reduce the assayed level of interferon in the subjectsblood to at most one third of a normal level of interferon, wherein thehuman subject has dementia.
 15. A method of preventing a disease of thecentral nervous system of a mammalian subject which comprisesadministering to a mammalian subject an amount of an interferonantagonist which blocks the action of an interferon receptor that iseffective to reduce the assayed level of interferon in the subjectsblood to at most one third of a normal level of interferon, wherein thesubject is at increased risk to accumulate amyloid in the centralnervous system.
 16. The method of claim 2 wherein the interferonantagonist will immunize the human against interferon.
 17. The method ofclaim 2 wherein the interferon antagonist has a different amino acidsequence compared to a wild-type interferon.